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Published ahead of print on March 30, 2006, doi:10.1164/rccm.200601-057OC
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American Journal of Respiratory and Critical Care Medicine Vol 174. pp. 15-20, (2006)
© 2006 American Thoracic Society
doi: 10.1164/rccm.200601-057OC

Original Article

Immunostimulatory Sequences Regulate Interferon-inducible Genes but not Allergic Airway Responses

Gail M. Gauvreau, Edith M. Hessel, Louis-Philippe Boulet, Robert L. Coffman and Paul M. O'Byrne

McMaster University, Hamilton, Ontario; Hopital Laval, Quebec City, Quebec, Canada; and Dynavax Technologies, Berkeley, California

Correspondence and requests for reprints should be addressed to Paul O'Byrne, M.B., HSC 3W10, McMaster University, 1200 Main Street West, Hamilton, ON, Canada, L8N 3Z5. E-mail: obyrnep{at}mcmaster.ca


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rationale: 1018 ISS is a synthetic oligonucleotide containing immunostimulatory CpG motifs. In animal studies, 1018 ISS effectively inhibited Th2-mediated lung inflammation, including eosinophil infiltration, and airway hyperresponsiveness.

Objectives: To evaluate whether 1018 ISS has activity in subjects with allergic asthma.

Methods: Forty subjects (n = 21, 1018 ISS; n = 19, placebo) were enrolled in a randomized, double-blind, placebo-controlled, parallel-group study to examine safety, pharmacologic activity, and efficacy of 1018 ISS on allergen-induced airway responses. Subjects received 36 mg of 1018 ISS or placebo by nebulization weekly for 4 wk.

Measurements: Allergen inhalation challenge was performed 24 h after the 2nd and 4th doses to measure the early and late fall in FEV1. Sputum cells and peripheral blood mononuclear cells were collected before and after dosing, and gene expression was measured by quantitative polymerase chain reaction.

Main Results: Treatment with 1018 ISS significantly increased expression of interferon (IFN)-{gamma} and IFN-inducible genes, such as IFN-{gamma}–inducible 10 kD protein (IP10), monokine induced by IFN-{gamma} (MIG), IFN-stimulated gene (ISG)-54, monocyte chemotactic protein (MCP)-1, and MCP-2 from cells collected postdose (p < 0.05). There was no attenuation of the early or late decrease in FEV1 after 1018 ISS compared with placebo, nor a reduction in allergen-induced sputum eosinophils or Th2-related gene expression measured in sputum cells.

Conclusions: This study demonstrated that 1018 ISS is safe and pharmacologically active in the respiratory tract of asthmatics but, at this dose regimen, did not inhibit a fall in FEV1 or other key features of the response to inhaled allergen challenge. This suggests that induction of IFN and IFN-inducible genes alone is not sufficient to inhibit allergen-induced responses in asthmatic subjects.

Key Words: airway responses • allergen inhalation challenge • allergic asthma • gene expression, immunostimulatory CpG motifs

The incidence and prevalence rates of allergic diseases continue to rise, now reaching epidemic proportions in the developed world (1). Therapies for treatment of allergic asthma are, in most cases, directed toward improving symptoms by reducing inflammation, and reversing or inhibiting bronchospasm. Despite a variety of pharmaceutical options, cure of these patients remains elusive, as there is no effective control of the underlying immune Th1/Th2 imbalance that perpetuates allergic disorders. Thus, there is a need for new effective strategies to reverse the Th2-biased inflammation that is associated with allergic disease.

Immunostimulatory DNA sequences, originally identified in bacterial DNA, contain unmethylated CpG dimers (ISS) that can stimulate the immune system. Sequences such as 1018 ISS have a nuclease-resistant phosphorothioate modification to prevent immediate breakdown. ISS act primarily on antigen-presenting cells to increase antigen presentation, increase costimulatory molecule expression, and induce the expression of interferons (IFNs) and IFN-inducible proteins (2). Several ISS-based therapeutic strategies have proved highly effective for the induction of Th1-biased immune responses and the prevention of Th2-biased immune deviation (3–7). The current strategies include immunization with allergen mixed with ISS (8), allergen–ISS conjugates (3, 9), and immunomodulation with ISS alone (4, 10).

As an immunomodulatory agent administered alone, ISS have a rapid effect on the allergic airway symptoms in mouse and primate models of asthma, which includes inhibition of eosinophilic airway inflammation and airway hyperresponsiveness (4, 6, 7, 10, 11). This inhibitory effect in vivo is associated with induction of Th1 cytokines such as IFN-{gamma}, and an inhibition of the Th2 cytokines interleukin (IL)-4, IL-5, and IL-13 (4–7). In accord with murine studies, mononuclear cells, B cells, and T cells isolated from atopic donors respond to ISS by producing cytokines known to inhibit allergic immune deviation (2, 12–14). Taken together with the results of studies using animal models, these results proved a rationale for conducting clinical trials of ISS-based immunomodulation for the treatment of allergic asthma.

This study investigates the effect of ISS using a well-characterized model of allergen inhalation by atopic subjects with asthma (15–17). This model has been used extensively to investigate the effects of various therapies on established asthma through measurement of allergen-induced bronchoconstriction, airway eosinophilia, and airway hyperresponsiveness (18–23). We hypothesized that treatment with ISS would inhibit the allergen-induced responses compared with placebo through up-regulation of IFNs and IFN-inducible genes and suppression of Th2-related gene expression. Some of the results of this study have been previously reported in an abstract (24).


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects
Subjects between 18 and 55 yr old were recruited for the study. Inclusion criteria required subjects to be nonsmokers with stable, mild atopic asthma, free of other lung disease. Subjects were required to have FEV1 of greater than 70% of predicted, baseline methacholine PC20 (the provocative concentration of methacholine causing a 20% fall in FEV1) of less than 16 mg/ml. For detailed inclusion criteria, see the online supplement.

Study Design
This multicenter, double-blind, randomized, placebo-controlled, parallel-group study, compared 4-wk treatment with 36 mg 1018 ISS administered weekly by inhalation (24). The study was performed at McMaster University and Laval Hospital after approval by the respective ethics research boards and on obtaining informed consent from study subjects.

Subjects meeting the inclusion criteria along with the development of an early asthmatic response (EAR) (at least 20% fall in FEV1 within 2 h after allergen inhalation) and late asthmatic response (LAR) (at least 15% fall in FEV1 between 3 and 7 h after allergen inhalation) were randomized to either 1018 ISS or placebo, 1:1.

Randomized subjects underwent allergen challenges after the second and the fourth doses on 3 consecutive days (Figure 1). Day 1 consisted of predose measurements of peripheral blood, airway hyperresponsiveness, and sputum cells, and the first dose of study medication was administered. On Day 2, a 24-h postdose measurement of peripheral blood was followed by an allergen-inhalation challenge. Measurements of FEV1 were taken at regular intervals until 7 h after challenge and sputum cells were then collected. On Day 3, subjects underwent 24-h postallergen (48-h postdose) measurements of peripheral blood, airway hyperresponsiveness, and sputum cells. Serum IgE levels were measured before and 1 mo after the end of treatment. All subjects completing the study were 100% compliant with treatment.


Figure 1
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  		Figure 1. Study design. PBMC = peripheral blood mononuclear cells.

 
Interventions
See the online supplement for detailed description of interventions and laboratory methods.

Study Medication
The 1018 ISS was prepared by adding 0.5 ml of 90 mg/ml solution to 3.2 ml normal saline and transferring 3.0 ml (36 mg 1018 ISS) to the nebulizer cup, and then was administered by inhalation using a PariLC Star nebulizer (Pari Respiratory Equipment, Mississauga, ON, Canada). Normal saline was used for placebo. An estimated 20% of the actual dose was delivered to the lung.

Methacholine Inhalation Test
Methacholine inhalation challenge was performed as described by Cockcroft (25). The test was terminated when a fall in FEV1 of 20% of the baseline value occurred, and the methacholine PC20 was calculated.

Allergen Inhalation Challenge
Allergen challenge was performed as described by O'Byrne and colleagues (15), and the concentration of allergen extract (Omega Laboratories, Montreal, PQ, Canada) for inhalation was determined from a formula described by Cockcroft and coworkers (26). The EAR is the maximum percentage decrease in FEV1 within 2 h after allergen inhalation, and the LAR is the maximum percentage decrease in FEV1 between 3 and 7 h after allergen inhalation.

Sputum Analysis
Sputum was induced and processed using the method described by Pizzichini and coworkers (27). Differential cell counts were obtained and an aliquot of cells were lysed for quantitative polymerase chain reaction (PCR) measurements.

Blood Analysis
Blood for peripheral blood mononuclear cells (PBMCs) was collected into cell preparation tubes (CPTs) (BD, Mississauga, ON, Canada) and centrifuged. The buffy coat layer was collected, and cells were lysed for quantitative PCR measurements.

Quantitation of Gene Expression by Real-Time PCR
Total RNA was extracted from cells and converted into cDNA. PCRs were performed using a GeneAmp 5700 Sequence Detector (Applied BioSystems, Foster City, CA) and the intercalating dye SYBR green (Qiagen, Valencia, CA) to detect the PCR product at each cycle (each PCR run consists of 40 cycles). Cycle threshold values were compared among samples. If the cycle threshold values within a sample set obtained from one patient varied more than two cycles from the mean ubiquitin expression of that particular sample set, the cDNA dilution was adjusted. All data were expressed as fold-change from predose baseline. As mRNA was measured from mixed cell populations, changes may reflect the level of gene expression as well as shifts in cell populations.

Statistical Analysis
Summary statistics are expressed as mean and SEM. The primary efficacy analysis compared the change from pretreatment baseline in allergen-induced LAR between 1018 ISS and placebo after 4 wk of treatment. The secondary analyses compared the change from pretreatment baseline in allergen-induced LAR after 2 wk of treatment, and the change from pretreatment baseline in allergen-induced EAR, methacholine PC20, and sputum eosinophils. The secondary analyses also compared the treatment-induced change of mRNA levels in sputum cells and PBMCs after 2- and 4-wk treatment. Comparisons of changes within subjects were analyzed using an analysis of variance (ANOVA) model, and between-treatment groups were analyzed with a t test using SAS System version 8.2 (SAS Institute Inc., Cary, NC).

The sample size was calculated using a key distribution. With a population mean LAR of 23.5% (SD, 11.2%), 15 subjects would be needed in each treatment group to demonstrate a 45% reduction of the LAR with a power of 90% and type I error rate of 5%.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Participant Flow
Fifty-five subjects were recruited for the study between January and September 2003. Of these subjects, 15 did not meet eligibility criteria. The remaining 40 subjects were randomized 1:1 to receive either 1018 ISS (n = 21) or placebo (n = 19). One subject from the 1018 ISS group and two subjects from the placebo group did not complete the study because of scheduling difficulties (Figure 2). Follow-up was performed between March and December 2003. Demographics and airway responsiveness were similar between the two groups (Table 1).


Figure 2
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  		Figure 2. Subject flow chart. EAR = early asthmatic response; LAR = late asthmatic response; ITT = intention to treat.

 

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  		TABLE 1. BASELINE DEMOGRAPHICS OF INTENT-TO-TREAT STUDY POPULATION

 
There were no serious adverse events reported during this study, and no adverse event was considered to be related to the study medication. The most common adverse event was headache, which was reported by six subjects in the placebo group and six subjects in the 1018 ISS group. There was no significant effect of 1018 ISS on serum IgE levels measured 1 mo after the end of treatment, being 160 x 103 U/L in the placebo group and 235 x 103 U/ml in the 1018 ISS group (p > 0.05).

Allergen-induced Airway Responses
Allergen inhalation induced early and late airway bronchoconstriction (Figure 3), increased eosinophil influx into the airways at 7 and 24 h after allergen challenge (Figure 3), and increased airway hyperresponsiveness to methacholine in both groups (data not shown). These allergen-induced changes are expected in this subject population. Treatment with 1018 ISS had no effect on the primary endpoint variable, LAR, nor was there attenuation of the secondary endpoint variables of EAR, sputum eosinophils (Figure 3), or airway hyperresponsiveness in the 1018 ISS-treated group (Dose 4 postallergen: placebo = 0.48 mg/ml;1018 ISS = 0.58 mg/ml; p > 0.05).


Figure 3
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  		Figure 3. (A) Mean allergen-induced fall in FEV1 in placebo (dotted line) and 1018 ISS (solid line) groups after Dose 4. (B) Mean sputum eosinophils before and at 7 and 24 h after allergen challenge in placebo (open bars) and 1018 ISS (solid bars) groups after Dose 4. {ddagger}p < 0.05 compared with preallergen baseline.

 
Treatment with 1018 ISS Up-Regulates Expression of IFN-inducible Genes in Both PBMCs and Sputum Cells
The 1018 ISS treatment had a significant effect on the secondary endpoint variables of gene expression. Inhaled 1018 ISS significantly increased the expression of IFN-inducible genes IFN-{gamma}–inducible 10 kD protein (IP10), IFN-stimulated gene (ISG)-54, monokine induced by IFN-{gamma} (MIG), monocyte chemotactic protein (MCP)-1, and MCP-2 in PBMC samples collected 24 h after dosing (p < 0.05), which returned to baseline by 48 h after dosing (Figure 4). Cells isolated from sputum also demonstrated significantly increased expression of the IFN-inducible genes IP10, MIG, ISG-54, MCP-1, and MCP-2 measured 31 h after 1018 ISS dosing (p < 0.05); IP10, MIG, ISG-54, and MCP-2 remained elevated 48 h after dosing (Figure 4). Gene expression of IFN-{alpha} in sputum cells increased by 3.4- and 2.4-fold at 31 h after 1018 ISS Doses 2 and 4, respectively, compared with placebo, which increased 1.4- and 2.0-fold, respectively. However, this did not reach statistical significance, possibly due to the reported waning of IFN gene expression after 24 h (3). Cells isolated from sputum also demonstrated significantly increased expression of IFN-{gamma} at 31 h after 1018 ISS Dose 2 (p < 0.05) but not after 1018 ISS Dose 4, increasing by 4.7- and 2.1-fold, respectively, compared with placebo increasing 2.0- and 1.7-fold, respectively.


Figure 4
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  		Figure 4. Expression of IFN-inducible mRNA measured from PBMC collected 24 and 48 h after Dose 4, and from sputum cells collected 31 and 48 h after Dose 4 in placebo (open bars) and 1018 ISS (solid bars) groups. *p < 0.05 placebo compared with 1018 ISS.

 
Expression of Th2-related Genes in Sputum Cells
Sputum contains a variety of cells, including high numbers of eosinophils and metachromatic cells. The percentage of T lymphocytes, however, is rather small or even undetectable in some patients (28). All of these cells are capable of making various cytokines and chemokines that are considered Th2 related, such as IL-4, IL-5, and thymus and activation–regulated chemokine (TARC). In this study, there was an increase in the expression of the Th2-related genes IL-4, IL-5, and TARC at 7 h after allergen challenge in the placebo group (p < 0.05), with IL-4 and IL-5 having the highest induction of approximately 25- and 40-fold increase, respectively, over preallergen levels. The expression of chemokine receptor 3 (CCR3), which is expressed on both eosinophils and a subset of Th2 cells, was also significantly increased after allergen challenge in placebo-treated patients (p < 0.05). All Th2-related gene expression of sputum cells approached baseline levels 24 h after challenge. Inhalation of 1018 ISS did not inhibit this allergen-induced Th2-related gene expression measured in sputum cells (p > 0.05; Figure 5).


Figure 5
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  		Figure 5. Expression of Th2-related gene expression measured from sputum cells collected 31 and 48 h after Dose 4 in placebo (open bars) and 1018 ISS (solid bars) groups. {ddagger}p < 0.05 compared with preallergen baseline.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The increasing incidence of allergic disease in developed countries coincides with progress in the area of infection control. It follows that a lack of exposure to bacterial products could conceivably result in a shift of the Th1/Th2 balance of the immune system leading to Th2 predominance. Data in murine and human systems have demonstrated that CpG-containing immunostimulatory DNA sequences enhance Th1 responses (12, 29, 30) and concomitantly inhibit preexisting Th2 responses, which are activities predicted to moderate a Th2-dominated allergic state. In a recent clinical trial, administration of 1018 ISS conjugated to the sensitizing allergen significantly reduced allergen-induced nasal inflammation in ragweed allergic individuals (31), providing evidence that rebalancing of Th2 to Th1 responses by ISS may be useful for treatment of Th2-driven allergic diseases. Because patients with asthma can be sensitized to multiple antigenic stimuli, administration of 1018 ISS conjugated to each sensitizing antigen is not a practical approach. An alternative therapeutic approach is to deliver 1018 ISS directly to the respiratory tract, the anatomic compartment chronically exposed to multiple offending allergens. The current study was designed as an initial exploration of this approach in subjects with asthma.

Administration of 1018 ISS by inhalation was found to be safe and well tolerated in this group of subjects with mild intermittent asthma. There was no worsening of asthma reported by subjects receiving 1018 ISS during this trial, despite the observed stimulation of a Th1 response commonly observed after respiratory virus infection. This suggests that, unlike viral infection, administration of 1018 ISS does not lead to exacerbation of asthma.

The results from this study demonstrate that inhalation of 1018 ISS was pharmacologically active, as shown by the induction of a set of IFN-regulated genes in peripheral blood and sputum cells. IFN-inducible genes remained elevated at 48 h after dosing in sputum cells, but returned to baseline by 48 h in PBMC. We did not observe increased levels of IFN-{alpha} or IFN-{gamma} mRNA in PBMCs at 24 h after inhalation, in accord with in vitro experiments showing that 1018 ISS–induced gene expression of IFN-{alpha} and IFN-{gamma} in PBMCs occurs much earlier than 24 h (29).

Despite induction of IFN and IFN-inducible genes during the time of allergen challenge, we did not observe any inhibition of the allergen-induced bronchoconstriction, hyperresponsiveness, or sputum-derived eosinophils. This suggests that induction of IFN and IFN-inducible genes alone is not sufficient to inhibit allergen-induced responses in subjects with asthma. A plausible explanation for a lack of effect is likely due to incomplete inhibition of the several types of immune cells involved in the allergic response, due to insufficient administration of 1018 ISS or insufficient treatment. Four weeks may not be long enough to induce immunotherapy in human atopic disease.

Because this was the first study to deliver 1018 ISS to subjects with asthma by inhalation, there had been no previous 1018 ISS inhalational studies to determine optimal dosing. The dose applied in this study was chosen based on results of an earlier phase I study, where aerosol administration of 36.0 mg 1018 ISS administered weekly for 4 wk was shown to be safe and well tolerated, and associated with the highest induction of genes considered as relevant surrogate markers of activity and potency of 1018 ISS in the respiratory tract. In the current study, 1018 ISS doses or route of administration may have been inappropriate or insufficient in the face of persistent allergen-induced eosinophilia and persistent Th2 cytokine expression. Given the dose-sensitive nature of ISS, providing suboptimal stimulation at lower concentrations but also exhibiting negative regulatory signals at high concentrations (29), it is not known whether another dose of 1018 ISS would have been effective.

Dosing with 1018 ISS for 4 wk has been shown to effectively inhibit allergen-induced airway hyperresponsiveness and airway eosinophilia in murine models of asthma (4–7). However, results in mice are not necessarily predictive of results in humans due to differences in toll receptor immune activation. ISS specifically target Toll-like receptor 9 (TLR-9), which is preferentially activated by CpG motifs that are different between humans and mice (32, 33). In addition, TLR-9 is expressed on a broader range of cells in mice, permitting ISS to directly target several immune functions, including the induction of Th1 and modification of antigen presentation.

In humans, however, TLR-9 expression has been reported to be restricted to B cells and plasmacytoid dendritic cells (34, 35). CpG triggering of TLR-9 induces plasmacytoid dendritic cells to produce high levels of IFN-{alpha} (8), which in turn induces cytokine production from other cell types—such as IFN-{gamma} production from natural killer cells—and modulates the actions of other immune cells. Although reports of indirect effects of ISS on human leukocytes include secretion of cytokines from human monocytes (36), production of IFN-{gamma} by human T cells following costimulation with ISS plus anti-CD3 or antigen (8, 30), and proliferation of T cells cocultured with CpG plus antigen (8), none of these reported functions demonstrate an effect of ISS alone on the response of memory T cells to antigenic stimulation. In the current study there was no inhibition of allergen-induced Th2 mRNA measured from inflammatory cells measured in the airways. Therapeutic benefit in subjects with asthma through administration of ISS alone may require extending the duration of treatment beyond the life cycle of existing memory T cells, which is estimated to be 60 to 70 population doublings (37, 38).

Development of ISS for treatment of allergic disease continues to be an exciting and promising area of study. Although the results from the current study have not shown an effect on allergen-induced responses in this model of allergic asthma, we did observe a pharmacologic effect through the induction of IFNs and IFN-inducible genes. The complex nature of ISS immune effects leading to regulation of Th2 pathways in humans will require further experimentation and refinement for future testing in asthma.


   Acknowledgments
 
The authors thank the following individuals whose expertise was critical for completion of this study: Rick Watson, Joanne Milot, and Francine Deschesnes carried out pulmonary function testing; Tara Strinich, Irene Babirad, Philippe Prince, and Marie-Eve Bouley prepared cells for analyses; and Kathryn S. Patton, Lolita Juarez, Tracy de la Cruz, Paul W. Sims, and Debbie Higgins completed all PCR measurements.


   FOOTNOTES
 
Supported by Dynavax Technologies.

This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Originally Published in Press as DOI: 10.1164/rccm.200601-057OC on March 30, 2006

Conflict of Interest Statement: G.M.G. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. E.M.H. is a full-time employee of Dynavax Technologies since January 2001 and holds stock and stock options. L.-P.B. received $150,000 in 2003 and 2004 from Dynavax Technologies in research grants. R.L.C. is a full-time employee of Dynavax Technologies since November 2000 and holds stock and stock options. P.M.O. is a consultant who sits on the advisory boards for AstraZeneca, Altana, GlaxoSmithKline, Topigen, Bristol-Myers Squibb, Roche, and Merck; he has also been a paid lecturer for these companies; he has held sponsored grants from Altana, AstraZeneca, Dynavax Technologies, GlaxoSmithKline, Biolipox, Ono, and Merck within the past 2 yr; he does not hold stock or stock options in any pharmaceutical company.

Received in original form January 13, 2006; accepted in final form March 24, 2006


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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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